Dr. Prashant Kumar Dr. Prashant Kumar Asst. Professor Asst. Professor Amity Institute of Virology & Immunology Amity Institute of Virology & Immunology Amity University Uttar Pradesh Amity University Uttar Pradesh Nucleic acid based multi-target approach for effective silencing of influenza A virus
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Dr. Prashant Kumar Asst. Professor Amity Institute of Virology & Immunology Amity University Uttar Pradesh Nucleic acid based multi-target approach for.
INFLUENZA Types of Influenza Virus ABC H1N1H3N2 (In Humans) Non Virulent H5N1 (In Birds)
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Dr. Prashant KumarDr. Prashant KumarAsst. ProfessorAsst. Professor
Amity Institute of Virology & ImmunologyAmity Institute of Virology & ImmunologyAmity University Uttar PradeshAmity University Uttar Pradesh
Nucleic acid based multi-target approach for effective silencing of influenza A virus
INFLUENZAINFLUENZA
One of the major Respiratory Virus Causative agent of one of the most
common disease i.e. common cold/ fever
Most easily transmissible disease- through aerosol
5’ NNNNNNNUX NNNNNNN YYYYYYYA YYYYYYY A CUG A A G AGU CG AU GC GC A G GU
3’ 5’3’
catalytic motif
target mRNA
Rz cleavage site
Designing of Hammer-head Ribozyme
Designing of siRNARules for selecting siRNA targets on mRNA sequences:
•Targets should be located 50-100 nt downstream of the start codon (ATG).•Search for sequence motif AA(N19)TT or NA(N21), or NAR(N17)YNN, where N is any nucleotide, R is purine (A, G) and Y is pyrimidine (C, U).•Target sequences should have a G+C content between 35-60%.•Avoid stretches of 4 or more nucleotide repeats.•Avoid 5'URT and 3'UTR, although siRNAs targeting UTRs have been shown to successfully induce gene silencing.•Avoid sequences that share a certain degree of homology with other related or unrelated genes.
Ribozymes for M gene of influenza A virus5’ CCGCAGGTG CTGCAGT
GGCGUCCA GACGUCA A CUG A A G AGU CG AU GC GC A G GU
3’ 5’3’
Rz-163
5’ GCCAGAATG CCACTGA CGGUCUUA GGUGACU A CUG A A G AGU CG AU GC GC A G GU
3’ 5’3’
Rz-106
Cloning of NP and M gene of influenza virus in pcDNA3
Co-transfection of HEK cells with siRNAs and cloned construct
In-vitro transcription of M gene construct
Analysis of the activity of Rz-163
Mg2+ (in mM)
809 nt
596 nt
213 nt
955 nt
623 nt
281 nt
596 nt
809 nt
213 nt
(c)(a)
1 2 1 2
Fig. 3
(b)0 5 10 25 50
1 2 3 4 5
Cleavage of target M1 RNA with Rz-163: Panel a: Autoradiograph Cleavage of target M1 RNA with Rz-163: Panel a: Autoradiograph showing full-length M1 RNA (lane 1) and cleaved fragments of showing full-length M1 RNA (lane 1) and cleaved fragments of target RNA with Rz (lane 2). Numbers on the left indicate size of target RNA with Rz (lane 2). Numbers on the left indicate size of RNA marker (cat # G3191, Promega). Panel b: lane 1 shows full RNA marker (cat # G3191, Promega). Panel b: lane 1 shows full length M1 RNA as before. Mutant Rz-163 failed to cleave the target length M1 RNA as before. Mutant Rz-163 failed to cleave the target RNA (lane 2). Panel c: The same transcript of M1 RNA was RNA (lane 2). Panel c: The same transcript of M1 RNA was subjected to cleavage by Rz-163 in the presence of varying amounts subjected to cleavage by Rz-163 in the presence of varying amounts of Mg2+ concentrations as shown on top ofof Mg2+ concentrations as shown on top of each lane. each lane.
Synthesis of novel Rz-chimeric-Synthesis of novel Rz-chimeric-siRNA constructs and its mutatntssiRNA constructs and its mutatnts
T7 promoter sequence sense loop anti-sense spacer Ribozyme
T7 promoter oligo in vitro transcription with T7 polymerase
5’ GCUAAAGACAA GACCAAU
CGAUUUCUGUU CUGGUUAACUGAUUCUUCAACUU
U
A
U C
G A
A
G
A3’ ACAGUGGA
C
A
U
G
G
UA
A
G
A
GUCC
CAGG
A GUG
spacer
5’ GCUAAAGACAA GACCAAU
CGAUUUCUGUU CUGGUUAACUGAUUCUUCAACUU 3’ ACAGUGGA
A
G
A
GUCC
CAGG
A GUG
spacerC
A
U
G
U
U A
U
A
U C
G A
A
G
A
5’ GCUCAGGACAU GAACAAU
CGAGUCCUGUA CUUGUUAACUGAUUCUUCAACUU 3’ ACAGUGGAC
A
U
G
G
UA
spacer
U
A
U C
G A
A
G
A
A
G
A
GUCC
CAGG
A GUG
5’ GCUCAGGACAU GAACAAU
CGAGUCCUGUA CUUGUUAACUGAUUCUUCAACUU 3’ ACAGUGGA
spacer
U
A
U C
G A
A
G
A
A
G
A
GUCC
CAGG
A GUG
C
A
U
G
U
U A
Fig. 2
(a)
(b) (c)
(d) (e)
wt-siRNA-wt-Rz mt-siRNA-wt-Rz
mt-siRNA-mt-Rzwt-siRNA-mt-Rz
In vitroIn vitro synthesized chimeric constructs (50ng) were incubated with synthesized chimeric constructs (50ng) were incubated with cytoplasmic protein extract (20 µg) for the period of 5 min at room cytoplasmic protein extract (20 µg) for the period of 5 min at room temperature and cleavage products were analyzed by gel temperature and cleavage products were analyzed by gel electrophoresis. Lane 1,3,5 and 7 represents uncleaved product and electrophoresis. Lane 1,3,5 and 7 represents uncleaved product and lane 2,4,6 and 8 represents cleaved product of 50 and 45 nucleotides.lane 2,4,6 and 8 represents cleaved product of 50 and 45 nucleotides.
MDCK cells grown to 70% confluencyMDCK cells grown to 70% confluency
co-transfection with 500 ng of M1-pcDNA3 co-transfection with 500 ng of M1-pcDNA3 plasmid DNA and various concentrations ranging plasmid DNA and various concentrations ranging from 1.5 to 5g of siRNA-chimeric-Rz constructsfrom 1.5 to 5g of siRNA-chimeric-Rz constructs
Total RNA isolation at 24h post transfectionTotal RNA isolation at 24h post transfection
RT PCR analysis with 1µg RNART PCR analysis with 1µg RNA
Real Time PCR analysis with 20ng RNAReal Time PCR analysis with 20ng RNA
Ribonuclease Protection Assay with 5µgRNA Ribonuclease Protection Assay with 5µgRNA
1 2 3GAPDH
M1 (759 bp)
siRNA-chimeric –Rz construct
No construct 1.5 µg 5.0 µg
M1 (759 bp)
1 2 3 4
GAPDH
No construct
mt-si-wt-Rz
wt-si-mt-Rz
wt-si-wt-Rz
chimeric constructs
C 1 2 3 4 5
mt-si-mt-Rz
wt-si-mt-Rz
wt-si-wt-Rz
constructs mt-si-wt-Rz- -
M1 RNA
- + + + + +Yeast t-RNA + - --
-
- -RNAse
- + + + + +
M1 specific probe + + + + + +
(a)
Fig. 4
Rel
ativ
e M
1 R
NA
ex
pres
sion
(%)
Rel
ativ
e M
1 R
NA
expr
essi
on (%
)R
elat
ive
M1
RN
A
expr
essi
on (%
)
(b)
(c)
0
20
40
60
80
100
120
1 2 3
0
20
40
60
80
100
120
1 2 3 4
0
20
40
60
80
100
120
1 2 3 4 5
Intracellular reduction in expression of M1 RNA with chimeric Intracellular reduction in expression of M1 RNA with chimeric constructs in MDCK cells: RNA level in MDCK cells were shown constructs in MDCK cells: RNA level in MDCK cells were shown by RT PCR and RPAby RT PCR and RPA.
Real Time RT PCR to show the modulation of expression level of M1 Real Time RT PCR to show the modulation of expression level of M1 gene of influenza virus in presence of various siRNA-Rz constructs: gene of influenza virus in presence of various siRNA-Rz constructs: SYBR Green based real time RT PCR was performed in triplicate and the SYBR Green based real time RT PCR was performed in triplicate and the level of M1 RNA in various experiments was compared by calculating level of M1 RNA in various experiments was compared by calculating ∆∆Ct value for each experiment. The RNA level of M1 in cells ∆∆Ct value for each experiment. The RNA level of M1 in cells transfected with only M1 clone was taken as the reference whose value transfected with only M1 clone was taken as the reference whose value was “1” and RNA levels in all other transfected cells were compared was “1” and RNA levels in all other transfected cells were compared with itwith it.
MDCK cells grown to a confluency of 70-80% in 6 MDCK cells grown to a confluency of 70-80% in 6 well platewell plate
Transfection with 5 µg of siRNA-Rz constructTransfection with 5 µg of siRNA-Rz construct
Infection of the transfected cells with inffluenza A Infection of the transfected cells with inffluenza A virus (A/PR/8/34) at 24h post transfectionvirus (A/PR/8/34) at 24h post transfection
FACS analysis of the infected cells at 10h post FACS analysis of the infected cells at 10h post infection for viral protein expression using M infection for viral protein expression using M
protein specific antibodiesprotein specific antibodies
Another group of cells observed for the cytopathic Another group of cells observed for the cytopathic effectseffects
Wild-type mutant constructs show inhibition of virus-specific immuno-fluorescence: Wild-type mutant constructs show inhibition of virus-specific immuno-fluorescence: Protection against Influenza A/PR8/34 virus challenge using chimeric constructs was Protection against Influenza A/PR8/34 virus challenge using chimeric constructs was studied using MDCK cells. The various siRNA-chimeric-Rz constructs (5µg/ml) were studied using MDCK cells. The various siRNA-chimeric-Rz constructs (5µg/ml) were transfected as indicated at the top of each panel and then challenged with a fixed dose transfected as indicated at the top of each panel and then challenged with a fixed dose of virus (MOI of 0.1). The cells were harvested 10 hours post infection and viral of virus (MOI of 0.1). The cells were harvested 10 hours post infection and viral protein levels were monitored by FACS analysis. Panels a to f represent the type of protein levels were monitored by FACS analysis. Panels a to f represent the type of constructs used for transfection and the levels were monitored by FACS analysis constructs used for transfection and the levels were monitored by FACS analysis
Treatment of cell line with chimeric constructs inhibits the multiplication of Treatment of cell line with chimeric constructs inhibits the multiplication of virus (infected at a MOI of 0.1) and reduces its cytopathic effect (CPE). virus (infected at a MOI of 0.1) and reduces its cytopathic effect (CPE).